Patients and controls
A total of 32 pregnant women who underwent complete curettage of uterine cavity in Jiangxi Maternal and Child Health Hospital from January 2020 to December 2020 were enrolled into the case group, and 32 healthy women who voluntarily requested for induced abortion due to unintended pregnancy during the corresponding gestational period were enrolled into the control group. In the diagnosis of missed abortion, transvaginal ultrasonography is primarily relied upon to detect empty gestational sacs or embryos/fetuses without cardiovascular beats. All included pregnant women had been screened for fetal chromosomal abnormalities, endocrine diseases, anatomical abnormalities, infections, immune diseases, trauma and medical diseases. All participants were also within 6-10 weeks of gestation and had no history of adverse pregnancy. Villi and decidua specimens were collected during vacuum aspiration under intravenous anesthesia. All samples were immediately processed in liquid nitrogen and then stored in a -80℃ refrigerator. Ten cases of villi and decidua in the EMA group and the control group, respectively, were selected for PRM-based targeted proteomic analysis (see Table1 for clinical baseline characteristics of the two groups). Based on the PRM results, western blotting and immunohistochemistry were performed on another 22 cases of villi from the EMA group and the control group. Compared with the control group, The clinical parameters of another 22 cases also showed no significant differences in maternal age,BMI, pregnancy duration,gravidity, parity and induced abortion. This study was supported by the Ethics Committee of Jiangxi Maternal and Child Health Hospital. All the studies provided informed consent.
LC-PRM detection and analysis
The total protein from villi and decidua were respectively extracted according to the instructions of the protein extraction kit, protein quantification was carried out using the BCA method, the samples were then aliquoted and stored in a -80°C refrigerator, afterward, 200ug were taken out of each sample for enzymatic hydrolysis by filter-assisted sample preparation (FASP), the peptides were desalinized and lyophilized after enzymatic hydrolysis, after which they were reconstituted with 0.1% formic acid, and had its concentration determined with OD280.
According to the pre-experimental results, 42 target peptides of 16 types of target proteins were given PRM quantitative analysis. The peptide information suitable for PRM analysis was imported into Xcalibur to configure the PRM method settings. About 1ug of peptides were taken from each sample and mixed with 20fmol of standard peptides for detection. The HPLC system was adopted for chromatographic fractionation. Buffer: A solution: 0.1% formic acid aqueous solution, B solution: 0.1% formic acid acetonitrile aqueous solution (acetonitrile of 84%). The column was equilibrated with 95% liquid A. The samples were fed into a chromatographic column for gradient separation at a flow rate of 300 nl/min.
The samples after HPLC separation were given PRM mass spectrum analysis using a Q-Exactive HF mass spectrometer (Thermo Scientific). The duration of the analysis was 60 min, and the detection mode was set to positive ion. All 40 samples received PRM detection respectively. In the end, the PRM raw data were analyzed in Skyline 3.5.0. The experimental procedure was following (refer to Fig. 1).
Western blotting validation
20ug of the extracted villi protein sample was separated by SDS-PAGE, then transferred to PVDF membrane and sealed with 5% defatted milk powder for 1 hour. The membrane was incubated with diluted primary antibodies overnight at 4℃. The membrane was incubated with secondary antibodies for 1 hour. The HRP signal was detected using hypersensitive ECL chemical reagent. The target protein bands were analyzed in Image J.
Human HSP90ab1 antibody(Boster,Wuhan,China,no.BM4191,1:2000 dilution), human HSPD1 antibody(Boster,Wuhan,China,no.M01280-3,1:2000 dilution) and human HSPAB13 antibody(proteintech,Wuhan,China,no.12667-2-AP,1:2000 dilution) were used as the primary antibody for Western blot analysis. The membrane were also incubated with anti-GAPDH antibody(ZSGB-BIO,no.TA-08,1:10000 dilution) to verify equal protein loading.
Immunohistochemistry validation
The paraffin-embedded villi tissue sections were dewaxed and dehydrated as per routine practice. Citric acid antigen repair buffer was used for antigen repair. In order to block endogenous peroxidase, the slices were incubated in 3% H2O2 at room temperature for 25min.The tissue sections were sealed with 3% BSA at room temperature for 30min and then incubated 50 μl of diluted primary antibodies overnight at 4 ° C. The primary antibody was anti-HSP90AB1(Boster, Wuhan, China, no.BM4191,1:200dilution), anti-HSPD1(Boster,Wuhan,China,no.M012803,1:200dilution)and anti-HSPA13(proteintech,Wuhan,China,no.12667-2-AP,1:200 dilution). The slices were incubated with the secondary antibodies of the corresponding species of the primary antibody at room temperature for 50min. Finally, fresh DAB display solution was added to the slices, and the color developing time was controlled under the microscope, and the positive color was brownish yellow. Two pathologists blinded to clinical and molecular data analyzed all sections,using modified H-scores[(percentage of weak intensity area ×1)+(percentage of moderate intensity area ×2)+(percentage of strong intensity area ×3)]. H-score is the value between 0 and 300, and the value is the larger , the comprehensive positive intensity is the stronger [12] .
Statistical analysis
Spss20.0(SPSS, Inc., Chicago, IL, USA) software was used for statistical analysis, and all data were expressed as mean ± standard deviation (SD). Student's T-test was used for comparison of quantitative data between the two groups, and P < 0.05 was considered statistically significant. Enumeration data is chi-square test. All experiments were repeated three times.